Mitochondrial haplotype variation and phylogeography of Iberian brown trout populations

The biogeographical distribution of brown trout mitochondrial DNA haplotypes throughout the Iberian Peninsula was established by polymerase chain reaction-restriction fragment polymorphism analysis. The study of 507 specimens from 58 localities representing eight widely separated Atlantic-slope (north and west Iberian coasts) and six Mediterranean drainage systems served to identify five main groups of mitochondrial haplotypes: (i) haplotypes corresponding to non-native, hatchery-reared brown trout that were widely distributed but also found in wild populations of northern Spain (Cantabrian slope); (ii) a widespread Atlantic haplotype group; (iii) a haplotype restricted to the Duero Basin; (iv) a haplotype shown by southern Iberian populations; and (v) a Mediterranean haplotype. The Iberian distribution of these haplotypes reflects both the current fishery management policy of introducing non-native brown trout, and Messinian palaeobiogeography. Our findings complement and extend previous allozyme studies on Iberian brown trout and improve present knowledge of glacial refugia and postglacial movement of brown trout lineages.     

Maintenance of an endemic lineage of brown trout (Salmo trutta) within the Duero river basin

The Iberian Peninsula contains diverse populations of freshwater fish, with major river basins comprising differentiated biogeographic units. The Duero River flows through the North‐Western Iberian Peninsula and is one of the most important rivers within the Iberian glacial refuge. Brown trout (Salmo trutta) populate this whole basin, and studies using both allozyme and microsatellite loci have detected a geographically sorted distribution of genetic variation in this species. In this work, sequences of the mitochondrial control region obtained from 299 brown trout from the Duero River were compared with other Iberian and European datasets. Two differentiated haplotype groups were detected inside the Duero River basin. One of them was related to the Atlantic (AT) lineage that is present in Northern European populations, whereas the other comprised an unique group that was restricted to the inner region of the basin. The amount of divergence of this Duero group from the other brown trout populations studied is consistent with a new trout lineage (Duero, DU) that is endemic to this river basin and that diverged from other Atlantic populations during the Pleistocene. The distribution of the DU and AT quaternary lineages in the Duero River was consistent with the ichthyological pattern described in the basin that originated during the Miocene–Pliocene. Evidence of selective processes that favour the haplotypes of the DU lineage may explain this discrepancy.     

Genetic structure of two Turkish brown trout populations

The genetic structure of two brown trout Salmo trutta populations living in Lake Abant in Bolu and Üzüm River in Antalya was determined by examining 15 enzyme coding loci ( AAT, ADH, LDH, MDH, MEP, GPI, PGM and SOD ) using starch gel electrophoresis. Population specific mobilities were observed for the fixed alleles of LDH-B2, mMEP-2 and SOD-1 loci. Polymorphisms in sAA T-4, GPI-B2 loci were observed within the populations. Average heterozygosity of Abant and Antalya populations was 0.0358 and 0.0224 respectively. For LDH-C which is the post glaciation marker locus, the ancestral allele * 105 was found to be fixed in both of the populations. Nei's genetic distance between the two populations was 0.2507 which is the level of genetic distance often found between different species. This difference seems to be due to the presence of unique alleles in the LDH-B2, mMEP-2 and SOD-1 loci of the Abant population, indicating that the conservation of the Abant population and its heterozygosity is of prime importance.     

Genetic structure of northwestern Spanish brown trout (Salmo trutta L.) populations, differences between microsatellite and allozyme loci

Genetic variation in nine wild brown trout (Salmo trutta L.) populations was studied by means of allozyme and microsatellite markers. All brown trout populations were clearly separated into two clusters that represented the Sil and Duero basins. Although both markers revealed a strong genetic differentiation between basins, microsatellite loci resulted much more accurate when population structure at the intrabasin level was analysed. Also pairwise multilocus FST estimates and assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. The analysis of both markers provides new insights in defining the conservation units at this local area and confirms the existence of a recognized sub-lineage in the Duero basin. The management implications of these findings are discussed and changes in trout release activity are recommended to avoid mixing of trout gene pools mainly in the Sil basin.     

A Highly Polymorphic Plasma Protein Locus in Brown Trout (Salmo trutta L.) Populations from Portugal

Genetic polymorphism of an unidentified plasma protein (PX) is described for the first time in Salmo trutta (L.) by means of isoelectric focusing. The analysis of 414 individuals from different geographic origins in Portugal allowed the identification of nine alleles. Heterozygosity in natural populations is generally above 0.60, thus giving similar values to those reported for brown trout microsatellite loci. Substructuring of Portuguese brown trout is evident between northern and southern basins. Genetic affinities between the southernmost rivers and the hatchery stock were detected, suggesting the existence of recent stocking influences.     

High Ag-NOR-site variation associated to a secondary contact in brown trout from the Iberian Peninsula

The analysis of nucleolar organizer regions (NORs) using silver (Ag-) staining and in situ hybridization (ISH) in brown trout (Salmo trutta) from various river basins in the Iberian Peninsula revealed high variation in the number and location of NORs. A total of 17 different Ag-NOR sites were revealed in 10 different chromosome pairs. Three different Ag-NOR patterns clustered by river basins and strongly associated to the internal transcribed spacer 1 (ITS1) variation were detected. The main variability in NOR-sites was found in a secondary contact between two divergent lineages of brown trouts at Duero basin. Our results confirmed the abrupt break in the spatial distribution of genetic variation of brown trout populations previously reported at Duero basin. We hypothesize that NOR-site variation might be a consequence of hybridization between divergent lineages of brown trouts and that NORs could play a major role in the maintenance of a hybrid zone in Duero basin via post-zygotic isolation mechanisms.     

Genetic variation and migration pathways of maritime pine (Pinus pinaster Ait) in the Iberian peninsula

The genetic variability and migration pathways of Pinus pinaster after glaciation in the Iberian peninsula was studied by means of 18 loci from 12 natural populations of the species. The analysis showed the existence of three groups of populations with different levels of diversity and patterns of recolonization. The southern Iberian group displays a high level of diversity, with a stepping-stone model of variation. The presence of rare alleles in this group and their position in the phylogenetic tree suggest the existence of refugia during glaciations in this zone. The eastern Iberian group also has high levels of diversity but is clearly separate from the first group based on their genetic distances. The Atlantic group displays a low level of diversity that could be interpreted as a rapid recolonization of the entire area by the Eastern group that has not yet developed to a divergence in this area. The southern Iberian range is indicated to be the dispersal centre of the species after the last glaciation. Received: 15 February 1999 / Accepted: 30 April 1999     

Population-genetic structure of Dolly varden (Salvelinus malma Walbaum) from Southeastern Sakhalin and Southern Kuril islands

The genetic structure of Dolly Varden (Salvelinus malma Walbaum) populations from six rivers of southeastern Sakhalin and six rivers of the Kuril Islands was examined using electrophoretic, analysis of 22 enzyme systems encoded by 45 loci. In all populations, mean heterozygosities (0.044-0.105), mean numbers of alleles per locus (1.15-1.52), and proportions of polymorphic loci (18.2-39.4%) were estimated for all loci that had clear resolution. Highly significant heterogeneity of allele frequencies was detected among populations of both Sakhalin and the Kuril islands. By means of cluster analysis and multidimentional scaling, all populations were divided into the Sakhalin and Kuril groups. Within the groups, no association of genetic similarity with geographic distance between populations was observed. Although the average genetic diversity was similar in these regions, the relative interpopulation diversity was almost three times higher in the Kuril (17.6%) than in the Sakhalin (6.5%) groups. In all, the proportion of genetic diversity between regions (8.4%) was somewhat lower than the proportion of mean interpopulation diversity (11.3%). In small populations isolated from the sea, genetic variation was lower than in nonisolated populations. Allozyme differences between the northern and the southern forms of Dolly Varden are discussed.     

Exploring alternative models for sex-linked quantitative trait loci in outbred populations: application to an iberian x landrace pig intercross

We present a very flexible method that allows us to analyze X-linked quantitative trait loci (QTL) in crosses between outbred lines. The dosage compensation phenomenon is modeled explicitly in an identity-by-descent approach. A variety of models can be fitted, ranging from considering alternative fixed alleles within the founder breeds to a model where the only genetic variation is within breeds, as well as mixed models. Different genetic variances within each founder breed can be estimated. We illustrate the method with data from an F(2) cross between Iberian x Landrace pigs for intramuscular fat content and meat color component a*. The Iberian allele exhibited a strong overdominant effect for intramuscular fat in females. There was also limited evidence of one or more regions affecting color component a*. The analysis suggested that the QTL alleles were fixed in the Iberian founders, whereas there was some evidence of segregation in Landrace for the QTL affecting a* color component.     

Genetic link between Chaoshan and other Chinese Han populations: Evidence from HLA-A and HLA-B allele frequency distribution

The genetic polymorphism of HLA-A and HLA-B loci was investigated in 505 Chaoshanese using PCR-SSP method. Among the HLA-A alleles detected, A*11 (35.64%) was most frequent, followed by A*02 (31.78%). Of 34 HLA-B alleles tested, 30 were observed, in which B*60 (21.68%), B*46 (14.46%), and B*58 (10.69%) were highly predominant. Comparison was made with other nine Chinese Han ethnic groups covering the Mainland China, Taiwan, Hong Kong, and Singapore. The high frequent alleles found in Chaoshanese were also common in other Chinese groups compared though the frequency levels varied from group to group. The phylogenic tree analysis based on the HLA-A and -B allele frequencies of all the 10 Chinese ethnic groups revealed that Chaoshanese, while clustering in general with the southern China-related Han Chinese, had the highest affinity to the Mainland Minnanese, but separated distinctively from the northern Han Chinese. The study, however, was yet to confirm the hypothesis of the Central Plains Han origin of Chaoshanese. Interestingly, the alleles (B*46, B*38, and B*58) and the related haplotypes (A*02-B*46 and A*33-B*58) that are positively associated with nasopharyngeal carcinoma (NPC), a disease prevailing predominantly among southern Chinese, were always at much higher frequencies among southern Chinese than among northern Chinese, whereas A*31 and B*13, the two alleles with highly protective effects for NPC, and the associated haplotype A*30-B*13 were predominantly high in northern Chinese. The different genetic background between northern and southern China may explain, at least partially, the prevalence of NPC among southern Chinese.     

The effect of recurrent floods on genetic composition of marble trout populations

A changing global climate can threaten the diversity of species and ecosystems. We explore the consequences of catastrophic disturbances in determining the evolutionary and demographic histories of secluded marble trout populations in Slovenian streams subjected to weather extremes, in particular recurrent flash floods and debris flows causing massive mortalities. Using microsatellite data, a pattern of extreme genetic differentiation was found among populations (global F(ST) of 0.716), which exceeds the highest values reported in freshwater fish. All locations showed low levels of genetic diversity as evidenced by low heterozygosities and a mean of only 2 alleles per locus, with few or no rare alleles. Many loci showed a discontinuous allele distribution, with missing alleles across the allele size range, suggestive of a population contraction. Accordingly, bottleneck episodes were inferred for all samples with a reduction in population size of 3-4 orders of magnitude. The reduced level of genetic diversity observed in all populations implies a strong impact of genetic drift, and suggests that along with limited gene flow, genetic differentiation might have been exacerbated by recurrent mortalities likely caused by flash flood and debris flows. Due to its low evolutionary potential the species might fail to cope with an intensification and altered frequency of flash flood events predicted to occur with climate change.     

Allozyme diversity in brown trout (Salmo trutta) from Central Spain: Genetic consequences of restocking

1.  The brown trout ( Salmo trutta ) represents one of the main freshwater resources in Spain, but habitat alterations and overharvesting have contributed to the decline or disappearance of numerous natural populations. In addition, reinforcement programs of wild populations based on releases of hatchery reared fish of exogenous origin compromise the conservation of remnant native trout resources.
2.  We present allozymic data from Central Spain trout populations including stocked and unstocked populations. Although the levels of genetic variation observed were low and affected by hatchery releases (p = 18.23%, Ho= 3.39%), they were within the range observed in other European areas.
3.  The effective introduction of hatchery reared fish is genetically homogenising the populations in the studied area and disturbing the ancestral pattern of genetic variation that distinguishes the Tajo and Duero basins. Within the eight natural populations analysed, seven had alleles assigned to the foreign trout. The introgression in these populations, following the LDH-5 * 90 allele frequency, ranged between 2% and 29.4%, but those values are not in concordance with the respective stocking effort undertaken in each population. Moreover, the release of hatchery-reared fish does not solve the problems related to the reduced size of wild populations and their recruitment instability.     

Silencing of duplicate genes: a null allele polymorphism for lactate dehydrogenase in brown trout (Salmo trutta)

A previously described isozyme polymorphism at one of two skeletal muscle LdhA loci in brown trout is due to a null allele, Ldh1(n), producing no detectable catalytic activity. Homozygotes for this allele have approximately only 56% of the LDH activity in skeletal muscle relative to homozygotes for the active allele. The remaining activity results from enzyme subunits produced by other LDH loci. The Ldh1(n) allele is common and widespread throughout brown trout populations in Sweden and is also found in populations from Ireland. The persistence of duplicate gene expression for the LdhA loci in almost all salmonid species is best explained by natural selection against individuals containing null alleles. However, there is no indication of natural selection against brown trout with the Ldh1(n) allele: We suggest that the selection against individuals containing null alleles that is apparently responsible for the persistence of duplicate LdhA loci in salmonids occurs only under certain environmental conditions.      

Management units of brown trout from Galicia (NW: Spain) based on spatial genetic structure analysis

Population genetic structure approaches offer the possibility of defining management units in conservation activities of species. The genetic structure of the brown trout Salmo trutta in Galicia (NW Spain) was investigated by using microsatellites. We determined genetic variation across 10 microsatellite loci of 901 individuals from 30 geographical populations representing 18 river basins. The analysis of the spatial distribution of the genetic variation by using different methods clearly revealed strong genetic differentiation among two groups of populations living in the studied area. This result is concordant with previous work using allozymes and mtDNA markers, and confirms a secondary contact among two highly differentiated evolutionary lineages in Miño Basin. Although both lineages might be locally adapted, results suggest that they hybridize at the middle course of the river. The brown trout from the Upper Miño Basin belongs to the previously described Duero lineage, an Iberian endemism threatened by introgression with other Atlantic forms. The results support the recognition of the Upper Miño Basin as a particular biotic region in Galicia. This study illustrates how a multidisciplinary approach in spatial genetic analysis contributes to the delineation of conservation units as conspecific metapopulations.     

Fine-spotted brown trout: genetic aspects and the need for conservation

In Lake Svartavasstjønn on the Hardangervidda plateau, a fine-spotted variant of brown trout has been found. The spotted variability shows a classical Mendelian mode of inheritance. In this population genetic variability was found in LDH-5 *, MDH-3 * and MDH-3 *. The fast allele at LDH-5 * had a frequency of 0,913. This is remarkable, as this allele has been found in frequencies above 0.4 in only five out of 50 investigated populations in Norway. The genetic characteristics of brown trout from Lake Svartavasstjønn were compared with 11 other populations sampled from a broad geographical area of Norway. Among these was Lake Setningen, where fine-spotted individuals occur at low frequency. Using a cluster analysis based on eight loci found to be polymorphic in brown trout, we found no indication of a close relationship between the fine-spotted brown trout from Lake Svartavasstjønn and the population in Lake Setningen. During a field survey in 1990 we found that the reproduction of fine-spotted trout in Lake Svartavasstjønn had terminated. The fine-spotted trout represents a valuable but threatened genetic resource, and attempts are carried out to conserve it.     

The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER. Xviii. Clinal and Uniform Genetic Variation over Populations

It has been reported in the previous papers of this series that in the eastern United States and Japan there is a north-to-south cline of additive genetic variance of viability and that the amount of the additive genetic variance in the northern population can be explained by mutation-selection balance. To determine whether or not the difference in the genetic variation in northern and southern populations can be explained by the differences in mutation rate and/or effective population size, numerical calculations were made using population genetic parameters. In addition, the average heterozygosities of the northern and southern populations at ten of 19 polymorphic structural loci surveyed were estimated in relation to the cline of additive genetic variance of viability, and the following findings were obtained. (1) The changes in mutation rate and population size cannot simultaneously explain the difference in additive genetic variance and inbreeding decline between the northern and southern populations. Thus, the operation of some kind of balancing selection, most likely diversifying selection, was suggested to explain the observed excess of additive genetic variance. (2) Estimates of the average heterozygosities of the southern population were not significantly different from those of the northern population. Thus, it was strongly suggested that the excess of additive genetic variance in the southern population cannot be caused by structural loci, but by factors outside the structural loci, and that protein polymorphisms are selectively neutral or nearly neutral.     

The evolutionary history of the allopolyploid Squalius alburnoides (Cyprinidae) complex in the northern Iberian Peninsula

Understanding the population structure, population dynamics and processes that give rise to polyploidy and helps to maintain it is central to our knowledge of the evolution of asexual vertebrates. Previous studies revealed high genetic diversity and several reproductive pathways in the southern populations of the Squalius alburnoides hybrid complex. In contrast, lower genetic variability and the associated limited chance of introducing new genetic combinations may threaten the survival of the northern Mondego populations. We analysed the genetic diversity and structure of nine populations of S. alburnoides in the Iberian Peninsula using microsatellite loci to provide further insights on the evolutionary history of this complex. Special attention was given to the less-studied northern populations (Mondego and Douro basins). Marked population structure, a high frequency of private alleles and a high diversity of some biotypes in the Douro basin indicate that some northern populations may not be at high risk of extinction, contrary to what was expected. The genetic diversity found in the northern Douro populations contradicts the general trend of remarkable genetic impoverishment northwards that occurs in other species and regions. The results indicate the possible existence of a glacial refugium in the Rabaçal River, corroborating findings in other species of this region. Historical events seem to have affected the geographical patterns of genetic variability found among and within the northern and southern populations of this complex and contributed to different patterns of genome composition. Therefore, historical events might have a major role in the long-term persistence of some polyploid hybrid taxa.     

A low-density single nucleotide polymorphism panel for brown trout (Salmo trutta L.) suitable for exploring genetic diversity at a range of spatial scales

The rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo trutta L.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site-associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non-target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.     

Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.